(a) Field of the invention
The present invention relates to a two-dimensional scanning type optical microscope.
(b) Description of the prior art
Conventional ordinary microscopse are arranged so that the entire region of observation of the sample under observation is illuminated as uniformly as possible by a light coming from a light source and by an appropriate condenser lens, and that the image of the sample is enlarged by an objective lens to be observed through an ocular lens or to be photographed. However, owing to the illumination of the entire observation region, there has tended to often develop such phenomenon as flare. In spite of various efforts which have been paid to solve these inconveniences, it has been impossible to obtain logical limit of resolution, and moreover samples of low contrast have been difficult to observe. Also, in case of the observation of a phase object by relying on, for example, differential infringement technique, the contrast of the sample under observation is also low, and thus it has been very difficult to observe its details. Also, in case a microscopic examination is performed to observe a phase object by a special microscopic technique such as the contrasting technique or the differential interference technique, or in case an observation in a dark field is conducted, it has been necessary to use specific expensive optical parts designed for the exclusive use of these respective microscopic techniques.
Therefore, in order to solve the problem that the logical limit of resolution cannot be attained because of the occurrence of such a phenomenon as flare, which is one of the drawbacks of the conventional optical microscopes, there has been proposed a microscope of a spot light projection type. This device is arranged so that a sample requiring an observation is illuminated in a spot form by the light coming from a spot light source, and that the light transmitting through the sample or the light reflected at the illuminated sample is again focused in a spot form, and that the information about the density of the image is obtained by a detector provided with a pinhole opening. This is a system almost similar to KOANANAORA system which is a photometric technique employed at present in, for example, micro-densitometry. With this system alone, there is obtained only a density information of the spot which is illuminated by the spot light. Therefore, arrangement has been provided so that, by a mechanical raster scanning of the sample two-dimensionally in X-Y directions, and by thus forming, for observation, an image on a CRT which is synchronous with said raster scanning.
Description will be made below with respect to the above-mentioned microscope based on an example described in the U.S. Pat. No. 3,013,467 Specification. FIG. 1 is a diagrammatic illustration thereof. A spot light source is formed with a light source 1 and a pin-hole 2. This spot light source is focused as a spot on a sample 4 by an objective lens 3 which has been well adjusted of its aberration, to illuminate the sample 4. Furthermore, the spot light projected onto the samples 4 is focused again as a spot onto a pin-hole 6 by a condenser lens 5 which has been well adjusted of its aberration, and the thus-formed spot light is detected by a detector 7 via the pin-hole 6. On the other hand, by a driving circuit 8, the surface of the sample 4 is mechanically scanned two-dimensionally in X-Y directions in such a way as by raster scanning for television. By indicating the image signal delivered from the detector 7 on a CRT 9 of the storage type which is synchronized with the synchronous signal coming from the driving circuit 8, it is possible to observe the image of the sample 4.
Since, in this prior art, arrangement is provided as stated above so that the sample is illuminated by a spot light and that the signal therefrom is detected by a spot-form detector, there is obtained a good image having a reduced flare as compared with the usual detectors, and also the resolution is improved. However, because this prior art represents a system of scanning which is performed by mechanically moving the sample, there have been problems such as an inconvenience in its handling. For example, samples have been limited to those which are light in weight and small in size, and also non-fixable samples such as cultured specimens encased in a decicator used in a laboratory have not been able to be observed. Furthermore, this prior art system has been difficult to apply to such a technique as flow cytometry, designed to observe different specimens successively and continuously.
Next, with respect to another problem that the above-mentioned conventional optical microscope requires exclusive optical parts and takes time and labor in making their adjustment for performing, for example, a special microscopy, there has been proposed a technique to use two detectors. This technique, like the above-described example, also is arranged so as to illuminate a sample in a spot form, and to detect the light which has transmitted therethrough or the light reflected at the sample, and to mechanically scan the sample two-dimensionally in X-Y direction in order to form the image of the sample.
Description of this microscope will be made in further detail by giving reference to FIG. 2. A spot light source 10 is focused as a spot onto a sample 4 by an objective lens 3 which has been well adjusted of its aberration, and the light which has transmitted through the sample 4 is detected by detectors 11 and 12 which are arranged to sandwich the optical axis 14. On the other hand, the sample 4 is mechanically scanned two-dimensionally in X-Y directions by a driving circuit 8 as in the case of raster scanning in television. And, the signals delivered from these detectors 11 and 12 are added or subtracted by an adder-subtractor 13 to be rendered to an image signal. By indicating this image signal on a storage type CRT 9 which is synchronized with the synchronous signal delivered from the driving circuit 8, the image of the sample can be observed. In case the signals supplied from the two detectors 11 and 12 are added together, an ordinary bright field image can be observed, whereas in case these signals are subjected to subtraction, a differential phase image of the sample 4 can be observed.
Here, description will be made of the logic of formation of a differential phase image.
For the sake of simplicity, one dimensional image is considered. The intensity of an image due to a partial coherent focusing is indicated, in general, as follows. ##EQU1## wherein: T(m) represents Fourier conversion function of the transmittivity of an object; and
C(m;p) corresponds to the transfer function of the optical system. PA1 .lambda. represents the wavelength of the light. PA1 m=.lambda.fm.
When the sensitivity of the detectors are assumed to be D(.xi.), and when the pupil function of the optical system is assumed to be P(.xi.), then C(m;p) is indicated by: ##EQU2## wherein: f represents the focal distance of the system; and
Here, D(.xi.) is assumed to be that of split detectors, and the difference in the signals will be considered, and we get ##EQU3## wherein: a represents the radius of the pupil; and
In case the object has a weak contrast, it is only necessary to consider only C(m;p). Accordingly, unless the effect of diffraction is considered, there will be obtained such a relation as EQU C(m;p)=m+p.
At such a time, we let ##EQU4## and we will note that I(.lambda.) contains the differentiation information ##EQU5## of the phase .phi.(.lambda.). Since t(.lambda.) represents amplitude, it will be noted that, by dividing the difference, signal of the detectors by the sum signal (=t.sup.2 (intensity)) of the detectors, there is obtained the information concerning differential phase.
As will be noted, in the case of the above-described prior art example, there can be performed a special microscopy which is called differential image observation only by a changeover of the connection of a switch without requiring the replacement of parts and making their adjustment. However, because there are used two detectors and moreover because they are fixed, not only there has arisen the inconvenience that the orientation of differentiation cannot be freely altered, but also, since the system is of the type that the sample per se is mechanically moved for the purpose of scanning, there has been the drawback that the device is inconvenient to handle.
Now, there has been proposed, in such a scanning type microscope as described above, a method of performing a dark field microscope by collecting the light reflected at the sample 4 with optical fibers arranged around the sample and by leading the collected light to a detector as shown in FIG. 3. This method is arranged so that the light emitting from a spot light source 14 such as a laser is irradiated onto a sample 16 by an objective lens 15, and that the light 17 which scatters on the sample 16 is collected by a light collector 18 comprised of optical fibers which are appropriately arranged around either the sample 16 or the objective lens 15, and that the collected light is detected by a detector 19. According to this method, however, in case a dark filed microscopy is to be performed it is necessary to use an expensive optical fibers so that there is the problem that the device becomes costly.